Selective determination of thiolic proteins by Hydrophobic Interaction Chromatography coupled with on-line Cold Vapour Atomic Fluorescence Spectrometry

A new analytical method is proposed for determination and characterization of thiolic proteins, based on hydrophobic interaction chromatography (HIC) coupled on-line with cold vapour atomic fluorescence spectrometry (CVAFS). Thiolic groups are derivatized pre-column by p-hydroxymercurybenzoate (PHMB) and the derivatized proteins are separated on a TSKgel Ether-5PW column. Post-column on-line reaction of derivatized proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of protein bound PHMB to inorganic mercury, Hg(II), which is selectively detected by AFS after sodium borohydride reduction to Hg°. Under optimized conditions, on-line bromine treatment gives a 85 ± 2 % recovery of both free and protein-complexed PHMB in less than 2.5 s and at room temperature. Glyceraldehyde-3-phosphate dehydrogenase, aldolase, pyruvate kinase, trioso phosphate isomerase and phospho-glucose isomerase have been examined. Sensitivity and limit of detection of proteins depends on number of –SH groups reacting with PHMB and are in the range of 10-8 – 10-9 mol dm-3 with calibration curves spanning over four decades of concentration.